Additional strains were recovered and grown from the large set of isolates of bacterial strains collected from patients with both HIV infection and periodontal disease. These isolates were previously isolated and speciated at Virginia Polytechnic Institute (VPI), Blacksburg, Virginia. Most of these bacteria are facultative or obligate anaerobes, and many are difficult to grow. In some cases, adjustments of the growth media and repeated trials were needed to obtain adequate growth. Although the VPI collection of bacteria is large, certain species previously associated with periodontal disease were not represented. Therefore, additional species were obtained from private individuals and from the ATCC. It is considered important to have a large and representative collection of strains so that the enzymatic and cytotoxicity assays will have a reasonable probability of detecting new virulence factors. Assays for adenylate cyclase and ADP-ribosylation were completed on representative strains from approximately twelve species. No strains producing these activities were found. For several species, variations in growth medium were explored to mimic the in vivo growth environment and stimulate production of virulence factors. Media were varied by including addition of carbon dioxide and limiting the availability of iron. Assays for additional virulence factors have been developed. Agar plate media were optimized for detection of protease, hemolysin, iron chelators, and lysozyme resistance.